Name: modifying baculovirus expression vectors to produce secreted plant proteins in insect cells
Uploaded: 2018-08-20T15:00:00.000+00:00
Duration: 9 min 14 s
Description: Watch this Scientific Journal Video about Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells at JoVE.com

这项工作得到了来自北卡罗来纳州立大学 Guozhou 许的新的教员启动基金的支持。

注: 采用修饰表达载体的昆虫细胞/杆状病毒系统, 用于分泌植物蛋白的表达和结晶。
1. 用 GP67 信号肽对杆状病毒表达载体进行植物蛋白分泌表达的改进
<ol><li>合成一个 DNA 片段, 包含 5 ' BglII 切割站点13, GP67 分泌信号序列, 和一个多克隆网站与 NotI, BamHI, EcoRI, StuI, 贝里沙, SpeI, XbaI, PstI 和 XhoI (图 1A)。 注: 由于 BglII 和 BamHI 消化 dna 导致相同的粘合剂末端, 线性化的载体可以结扎到被消化的脱氧核糖核酸片断, 当退火的结扎序列的 BglII 和 BamHI 站点将被变异到不是裂解的序列无论是 BglII 或 BamHI14。</li>
	<li>消化4µg 的 dna 与 BglII 和 XhoI, 和4µg 的表达载体 dna BamHI 和 XhoI14。将每种限制酶的10个单位与1x 浓度反应缓冲器中的 DNA 混合 (50 毫米醋酸钾, 20 毫米三醋酸盐, 10 毫米醋酸镁, 100 µg/毫升 BSA, pH 7.9 在25°c) 和孵化混合物在37°c 为 4 h。<ol>
<li>用 dna 凝胶萃取试剂盒15纯化分离的 dna 片段和线性化的载体 dna。</li>
	</ol>
</li>
	<li>在含有 1x T4 DNA 连接酶反应缓冲器 (50 毫米三盐酸) 的 10-µL 结扎反应混合物中, 将100的线性化向量 DNA 与 400 ng 的被消化 dna 片段混合在一起; 10 毫米氯化镁2, 1 毫米 ATP, 和10毫米的德勤, pH 7.5 在25°c) 和5个单位的 T4 DNA连接酶.孵育反应混合物在16°c 为16小时。</li>
	<li>将结扎合剂的5µL 转化为100µL 的 DH5α合格细胞, 遵循标准的转换协议, 并选择在氨苄西林-LB 琼脂板16上产生的菌落。拿起 5-10 殖民地和生长每殖民地在2毫升的 LB 培养基包含100µg/毫升氨苄西林在 220 rpm 和37°c 在一个震动孵化器为 16 h。</li>
	<li>提取每个质粒 dna 与 dna miniprep 试剂盒17和确认克隆的 dna 测序与底漆 AGTATTTTACTGTTTTCGTAACAG。</li>
	<li>PCR-放大a.拟南芥基因片段编码残留物 30-642 从合成 TDR 基因构造 (向前底漆: CATG GCGGCCGC CTCAAGTTTTCACCTCAACTCTTGTC, 反向底漆: GC GGATCC CTA GTG GTG ATG GTG GTG GTG ACCGTCTATATCTGCATTTCCGGC)。在 dna 聚合酶反应缓冲器中放大30周期的 dna, 其中包含0.5 毫米 dNTP 混合, 0.2 µM 底漆, 0.1 µg 的模板 dna, 0.4 毫米氯化镁2和1反应单元的 dna 聚合酶。<ol>
<li>为 PCR 周期步骤, 设置最初变性在95°c 三十年代, 然后30周期变性在95°c 三十年代, 退火在55°c 为三十年代, 并且引伸在72°c 为 1 min, 以最后的引伸在72°c 为5分钟。</li>
	</ol>
</li>
	<li>用 NotI 和 BamHI 限制限制性酶对 PCR 片段和修饰载体进行消化。用线性化向量结扎基因片段。将 100 ng 的线性化矢量 DNA 与400的消化 dna 片段 (含 T4 dna 连接酶反应缓冲器和5单位 T4 dna 连接酶) 的µL 结扎反应混合物混合。孵育反应混合物在16°c 为16小时。</li>
	<li>将结扎合剂的5µL 转化为100µL 的 DH5α合格细胞, 遵循标准的转换协议, 并选择在氨苄西林-LB 琼脂板16上产生的菌落。通过 DNA 测序确认正确的克隆。</li>
</ol>2. 昆虫 Hemolin 信号肽用于植物蛋白分泌表达的杆状病毒表达载体的改进
<ol><li>合成一个 DNA 片段, 包含 5 ' BglII 切割站点13, 昆虫 hemolin 分泌信号序列, 和一个多克隆网站与 NotI, BamHI, EcoRI, StuI, 贝里沙, SpeI, XbaI, PstI 和 XhoI (图 1B)。</li>
	<li>消化4µg 的 dna 与 BglII 和 XhoI, 和4µg 的表达载体 dna BamHI 和 XhoI14。将每种限制酶的10个单位与1x 浓度反应缓冲器中的 DNA 混合 (50 毫米醋酸钾, 20 毫米三醋酸盐, 10 毫米醋酸镁, 100 µg/毫升 BSA, pH 7.9 在25°c) 和孵化混合物在37°c 为 4 h。<ol>
<li>用 dna 凝胶萃取试剂盒15纯化分离的 dna 片段和线性化的载体 dna。</li>
	</ol>
</li>
	<li>在含有 1x T4 DNA 连接酶反应缓冲器 (50 毫米三盐酸) 的 10-µL 结扎反应混合物中, 将100的线性化向量 DNA 与 400 ng 的被消化 dna 片段混合在一起; 10 毫米氯化镁2, 1 毫米 ATP, 和10毫米的德勤, pH 7.5 在25°c) 和5个单位的 T4 DNA连接酶.孵育反应混合物在16°c 为16小时。</li>
	<li>将结扎合剂的5µL 转化为100µL DH5α合格细胞, 并选择氨苄西林-LB 琼脂板上的菌落。拾起 5-10 殖民地并且生长每个殖民地在一个2毫升 LB 媒介包含100µg/毫升氨苄西林在 220 rpm 和37°c 在一个震动孵化器为 16 h。</li>
	<li>用 dna miniprep 试剂盒17提取质粒 dna, 并通过 AGTATTTTACTGTTTTCGTAACAG 的 dna 测序确认克隆。</li>
	<li>PCR-放大a. 芥花粉受体激酶 3 (PRK3) 基因片段编码残留物 20-237 从合成 PRK3 基因构造 (向前底漆: CATG GCGGCCGC CTACAAAACGTTAGTGAATCGGAACC, 反向底漆: CGC GGATCC CTA GTG GTG ATG GTG GTG GTGTGAAGGTTTCTCATCGCATTCTATATTC)。在 dna 聚合酶反应缓冲器中放大30周期的 dna, 其中包含0.5 毫米 dNTP 混合, 0.2 µM 底漆, 0.1 µg 的模板 dna, 0.4 毫米氯化镁2和1反应单元的 dna 聚合酶。<ol>
<li>为 PCR 周期步骤, 设置初始变性在95°c 三十年代, 然后30周期变性在95°c 三十年代, 退火在55°c 为三十年代, 并且延长在72°c 为三十年代在每个周期之内, 并且最后的引伸在72°c 为5分钟。</li>
	</ol>
</li>
	<li>用 NotI 和 BamHI 限制限制性酶对 PCR 片段和修饰载体进行消化。用线性化向量结扎基因片段。将 100 ng 的线性化矢量 DNA 与400的消化 dna 片段 (含 T4 dna 连接酶反应缓冲器和5单位 T4 dna 连接酶) 的µL 结扎反应混合物混合。孵育反应混合物在16°c 为16小时。</li>
	<li>将结扎合剂的5µL 转化为100µL 的 DH5α合格细胞, 遵循标准的转换协议, 并选择在氨苄西林-LB 琼脂板16上产生的菌落。通过 DNA 测序确认正确的克隆。</li>
</ol>3. 以重组蛋白表达盒为载体的杆状病毒结构的制作和扩增
<ol><li>利用构造质粒100的 DH10Bac, 在杆状病毒表达系统1的协议下, 转化40µL 的合格细胞。在37摄氏度的48小时内孵化转化板。</li>
	<li>从每个变换板上提取两个白色的菌落, 将每个菌落接种2毫升的 LB 培养基, 其中含有50µg 卡那霉素、7µg/毫升庆大霉素, 以及10µg/毫升四环素。遵循杆状病毒表达系统1的协议提取和验证 bacmid DNA。整除每个 DNA 在两个管与20µL 每一个, 并存储管在-20 摄氏度。 注意: 以下步骤需要无菌操作。</li>
	<li>在培养基中培养10% 胎牛血清 (血清) 和100µg/毫升 (或100国际单位/毫升) 青霉素-链霉素抗生素, 在27摄氏度至80% 井板的 Sf9 细胞。根据组织培养板上覆盖面积的百分比估计汇流的百分比。</li>
	<li>在两个无菌1.5 毫升离心管中准备以下解决方案。<ol>
<li>管 1: 每次转染, 稀释20µL bacmid DNA 成100µL unsupplemented Sf9 细胞培养基, 无抗生素。轻轻地把稀释后的管子的侧面混合。</li>
		<li>管 2: 对每转染, 稀释8µL 脂转染试剂成100µL unsupplemented Sf9 细胞培养基无抗生素。将稀释液彻底混合, 吹打上下6x。</li>
	</ol>
</li>
	<li>结合两个解决方案, 轻轻地混合在一起, 吹打慢慢向上和向下 3x, 并孵化他们在室温下 20-40 分钟。</li>
	<li>用3毫升的 unsupplemented Sf9 细胞培养培养基 Sf9 单层细胞 2x, 无抗生素。每次转染, 添加0.8 毫升的 unsupplemented Sf9 细胞培养基, 不含抗生素, 每管含有脂质 DNA 混合物。轻轻地将管子的内容混合在一起, 慢慢地吹打3x。从盘子中吸取洗涤介质, 然后用转染混合物覆盖样品。</li>
	<li>在添加转染混合物后, 在27摄氏度的5小时内孵化出所染的板。用完整的 Sf9 细胞培养基取代转染培养基, 其中含有10% 只血清和100µg/毫升 (或100国际单位/毫升) 青霉素-链霉素抗生素。在27摄氏度孵育转染板 4 d。</li>
	<li>收获和放大重组杆状病毒。<ol>
<li>在 4 d 孵化后, 在不育的15毫升圆锥管中收集感染板的上清。以 1010 x g为5分钟旋转上清, 去除细胞碎片。丢弃颗粒并将上清醒酒到干净的管子上, 将其贮存在摄氏4摄氏度, 长达1年。 注意: 这是 P0 病毒。它可以 aliquoted 和储存在-80 摄氏度, 在较长的时间内。</li>
		<li>为了放大每种重组病毒, 在150毫米的板块上生长一层 Sf9 细胞到80% 汇合。从盘子中吸取培养基, 将2毫升的 P0 病毒添加到附着在盘子上的细胞中, 在27摄氏度孵化器中孵化出1小时的平板。每隔15分钟摇动盘子, 将培养基与细胞混合。<ol>
<li>添加25毫升完整的恩典的媒体含有10% 血清和100µg/毫升 (或100国际单位/毫升) 青霉素-链霉素抗生素。在27摄氏度孵化器中孵化出 3 d 的盘子, 以获得 P1 通道病毒。</li>
		</ol>
</li>
		<li>在不育的50毫升圆锥管中收集被感染的盘子的上清, 并在 1010 x g处旋转5分钟以去除细胞碎片。醒酒到干净的管, 并保存在4摄氏度, 长达1年。 注: P1 病毒可以 aliquoted 和储存在-80 摄氏度, 较长一段时间。</li>
		<li>为了将病毒从 P1 扩大到 P2 通道, 在150毫米的盘子上长出一层 Sf9 细胞到90% 汇合处, 吸出盘子的培养基, 在盘子中加入2毫升 P1 病毒, 并在27摄氏度孵化器中孵化1小时。</li>
		<li>重复步骤3.8.2 和3.8.3 获取病毒的 P2 和 P3 通道。不要将 P3 病毒存储2月以上。</li>
	</ol>
</li>
</ol>4. 蛋白质表达, NiNTA 纯化和结晶
<ol><li>培养 1 L Hi5 昆虫细胞在文化媒介与100µg 或 ml (或100国际单位/毫升) 青霉素-链霉素抗生素到密度 2 x 106在 100 rpm 在27°c 孵化器振动筛。向下旋转 570 x g的细胞为5分钟, 醒酒上清, 并用重悬的细胞在20毫升的新鲜生产的 P3 病毒, 并保持在室温下1小时. 轻轻摇动旋转瓶, 并用重悬细胞1x 每15分钟。</li>
	<li>将细胞转移到1升的培养基上, 用100µg/毫升 (或100国际单位/毫升) 青霉素-链霉素抗生素, 培养细胞在 100 rpm 在27°c 孵化器振动筛为 72 h. 在 1010 x g的细胞中旋转5分钟, 然后收集上清。</li>
	<li>使用 ph 指示条将上清液的 ph 值调整为 8.0, 加入0.1 米 HCl 或0.1 米氢氧化钠, 并在最终浓度下添加捆绑缓冲器, 在 pH 8.0、100毫米氯化钠和5毫米咪唑中含有20毫米的三个缓冲器。添加一定数量的 NiNTA 树脂 (10-40 毫升) 根据估计产量的表达他标记的重组蛋白。<ol>
<li>在4°c 的1小时内, 将溶液与低速的磁搅拌混合在一起, 清洗树脂并洗脱标准 NiNTA 纯化协议18中的绑定蛋白。</li>
	</ol>
</li>
	<li>以纯化蛋白为离子交换和凝胶过滤层析, 以及其他蛋白质纯化方法, 产生均匀样品。 注: 对于 TDR 胞外区, 20 毫米三, pH 8, 100 毫米氯化钠被用作凝胶过滤缓冲器。对 PRK3 胞外区, 20 毫米双三, pH 6, 100 毫米氯化钠作为首选凝胶过滤缓冲剂。</li>
</ol>

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J., Wu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+method+for+inserting+specific+DNA+sequences+into+cloning+vehicles.">A general method for inserting specific DNA sequences into cloning vehicles.</a> Gene. 1, 81-92 (1976).</li><li>Xu, W., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Vaccine-Derived+Polioviruses,+Relevance+for+the+Global+Polio+Eradication+Initiative.">Isolation and Characterization of Vaccine-Derived Polioviruses, Relevance for the Global Polio Eradication Initiative.</a> Methods in Molecular Biology. 1387, 213-226 (2016).</li><li>Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+transformation+of+Escherichia+coli+with+plasmids.">Studies on transformation of Escherichia coli with plasmids.</a> Journal of Molecular Biology. 166, 557-580 (1983).</li><li>Zhang, S., Cahalan, M. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+heterologous+genes+in+E.+coli.">Expression of heterologous genes in E. coli.</a> Microbiological Sciences. 3, 28-31 (1986).</li><li>Kaur, J., Kumar, A., Kaur, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+optimization+of+heterologous+protein+expression+in+E.+coli:+Roadblocks+and+reinforcements.">Strategies for optimization of heterologous protein expression in E. coli: Roadblocks and reinforcements.</a> International Journal of BIological Macromolecules. 106, 803-822 (2018).</li><li>Guo, Y., Yang, F., Tang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Overview+of+Protein+Secretion+in+Yeast+and+Animal+Cells.">An Overview of Protein Secretion in Yeast and Animal Cells.</a> Methods in Molecular Biology. 1662, 1-17 (2017).</li><li>Blight, M. A., Chervaux, C., Holland, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+secretion+pathway+in+Escherichia+coli.">Protein secretion pathway in Escherichia coli.</a> Current Opinion in Biotechnology. 5, 468-474 (1994).</li><li>Idiris, A., Tohda, H., Kumagai, H., Takegawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+protein+secretion+in+yeast:+strategies+and+impact+on+protein+production.">Engineering of protein secretion in yeast: strategies and impact on protein production.</a> Applied Microbiology and Biotechnology. 86, 403-417 (2010).</li><li>Wormald, M. R., Dwek, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoproteins:+glycan+presentation+and+protein-fold+stability.">Glycoproteins: glycan presentation and protein-fold stability.</a> Structure. 7, R155-R160 (1999).</li><li>Geisler, C., Mabashi-Asazuma, H., Jarvis, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Overview+and+History+of+Glyco-Engineering+in+Insect+Expression+Systems.">An Overview and History of Glyco-Engineering in Insect Expression Systems.</a> Methods in Molecular Biology. 1321, 131-152 (2015).</li><li>Li, Z., Chakraborty, S., Xu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+crystallographic+studies+of+the+extracellular+domain+of+the+first+plant+ATP+receptor,+DORN1,+and+the+orthologous+protein+from+Camelina+sativa.">X-ray crystallographic studies of the extracellular domain of the first plant ATP receptor, DORN1, and the orthologous protein from Camelina sativa.</a> Acta Crystallographica Section F: Structural BIology and Crystallization Communications. 72, 782-787 (2016).</li><li>Aricescu, A. R., Owens, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+recombinant+glycoproteins+in+mammalian+cells:+towards+an+integrative+approach+to+structural+biology.">Expression of recombinant glycoproteins in mammalian cells: towards an integrative approach to structural biology.</a> Current Opinion in Structural Biology. 23, 345-356 (2013).</li><li>Reuter, L. J., Bailey, M. J., Joensuu, J. J., Ritala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale-up+of+hydrophobin-assisted+recombinant+protein+production+in+tobacco+BY-2+suspension+cells.">Scale-up of hydrophobin-assisted recombinant protein production in tobacco BY-2 suspension cells.</a> Plant Biotechnology Journal. 12, 402-410 (2014).</li><li>Dohi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+virus-mediated+expression+of+a+foreign+protein+in+suspension-cultured+plant+cells.">Inducible virus-mediated expression of a foreign protein in suspension-cultured plant cells.</a> Archives of Virology. 151, 1075-1084 (2006).</li></ol>

作者没有什么可透露的。

鉴于生物系统中数以千计的蛋白质的大小和稳定性的多样性, 研究实验室通常有经验来决定哪些异种表达系统必须选择用于表达特定的蛋白质。大肠杆菌表达系统往往是由于细菌生命周期短、培养基成本低、相对容易扩展19而导致蛋白质表达的首选。然而, 对于大小超过 60 kDa 的大真核蛋白的表达, 使用大肠杆菌系统往往导致不溶性蛋白在包涵体或聚集20<...

研究实验室有能力生产大量的均质重组蛋白, 用于生化和生物物理特征, 尤其是 x 射线晶体学研究。大肠杆菌、酵母、昆虫细胞、哺乳动物细胞、植物细胞等具有良好的外源表达系统, 其中杆状病毒介导的昆虫细胞表达系统是其中最常用的技术生产大量的结构折叠大型重组真核蛋白的蛋白质结晶1。
对杆状病毒表达系统的表达载体进行了设计, 以包含强多角体或 P10 启动子, 以产生高产量的重组细胞内蛋白2,3。为了制作重组杆状病毒, 该基因被克隆成含有杆状使用杆状多 nucleopolyhedroviral 基因组多角体 (polh) 轨迹的昆虫载体。然后对生成的构造进行排序, 验证其正确的开放阅读框架 (ORF)。通过转染过程将正确的构造引入宿主昆虫细胞。兴趣基因通过同源重组插入病毒基因组。这一事件导致重组病毒基因组的生产, 然后复制, 以产生重组发芽病毒粒子1。
在表达系统中最常用的昆虫细胞是 Sf9 和高五 (Hi5) 细胞。Sf9 细胞是 Sf21 蛹卵巢细胞的克隆分离物, Hi5 细胞是由父母夜蛾镍卵巢细胞系 TN-3684,5所衍生的克隆分离株。transfections、病毒扩增和斑块检测是在 Sf9 细胞上进行的, 而 Hi5 细胞通常被选择以产生更高数量的重组蛋白6。值得注意的是, Hi5 细胞由于其产生突变病毒的倾向而不适合于病毒后代的产生和扩增。传统上, 25-30 °c 的温度范围被认为对昆虫细胞的培养是有益的。然而, 据报道, 27-28 °c 是最佳的温度为昆虫细胞生长和传染7,8。
在基因之前引入一个强烈的信号序列是分泌蛋白的高表达所必需的。该信号序列将有效地引导转化后的重组蛋白转化为内质网的蛋白质分泌和转化后的修改必要的适当折叠和稳定3。信号肽序列, 如杆状病毒包络蛋白 GP64/67, 蜜蜂蜂毒等, 已被选择, 以促进分泌重组蛋白的表达在杆状病毒介导的表达系统3。GP67 的信号肽的引入, 以提高分泌重组蛋白的表达率, 与使用目标基因9的内在信号肽相比较。Hemolin 是大丝蛾Hyalophora cecropia的血淋巴蛋白, 引起细菌感染10。由于诱导表达水平较高, 该基因的信号肽序列可以用来调节杆状病毒-昆虫细胞系统中重组蛋白的分泌表达。
Tracheary 元素分化抑制因子受体 (TDR) 和花粉受体激酶 3 (PRK3) 都属于植物亮氨酸丰富的重复受体样激酶 (LRR RLK) 蛋白11,12 的家庭.为了研究植物受体蛋白家族的结构和功能, 以及促进其他植物分泌蛋白的结构和生化特性, 对杆状病毒-昆虫细胞表达系统进行了改进, 以提高蛋白质质量和产量。利用两种修饰表达载体在杆状病毒-昆虫细胞表达系统中成功地表达了 TDR 和 PRK3 的胞外域。TDR 和 PRK3 蛋白的胞外域均已结晶。本文报道了两种改进的杆状病毒表达载体的大量重组分泌植物蛋白的表达和纯化, 通过在启动子和多克隆之间加入 GP67 或 hemolin 信号序列。网站。

<table><tbody><tr><td>Incubator shaker</td><td>VWR</td><td>Model Excella E25</td><td>27 &lt;sup&gt;o&lt;/sup&gt;C</td></tr><tr><td>Incubator</td><td>VWR</td><td>Model 2005</td><td>27 &lt;sup&gt;o&lt;/sup&gt;C</td></tr><tr><td>Centrifuge</td><td>BECKMAN</td><td>Model J-6</td><td>with a swing bucket rotor</td></tr><tr><td>Herculase II Fusion DNA Polymerase</td><td>Agilent</td><td>600677-51</td><td>For PCR amplification of DNA</td></tr><tr><td>Thermal Cycler</td><td>BIO-RAD</td><td>Model C1000 Touch</td><td>For PCR amplification of DNA</td></tr><tr><td>Incubator shaker</td><td>New Brunswick Scientific</td><td>Model I 24</td><td>For growing baceria culture</td></tr><tr><td>Customer DNA synthesis</td><td>GENSCRIPT</td><td></td><td></td></tr><tr><td>BamHI (HF)</td><td>New England Biolabs</td><td>R3136S</td><td>Restriction Endonuclease</td></tr><tr><td>BglII</td><td>New England Biolabs</td><td>R0144S</td><td>Restriction Endonuclease</td></tr><tr><td>NotI (HF)</td><td>New England Biolabs</td><td>R3189S</td><td>Restriction Endonuclease</td></tr><tr><td>XhoI</td><td>New England Biolabs</td><td>R0146S</td><td>Restriction Endonuclease</td></tr><tr><td>T4 DNA ligase</td><td>New England Biolabs</td><td>M0202T</td><td>DNA ligation</td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td>DNA purification from Agorase gel</td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27104</td><td>Plasmid DNA purification from bacteria culture</td></tr><tr><td>Agarose</td><td>Thermo Fisher Scientific</td><td>15510-019</td><td>For DNA gel electropherosis</td></tr><tr><td>MAX Efficiency DH5&amp;alpha; competen cell</td><td>Invitrogen</td><td>18-258-012</td><td>For transformation of DNA ligation mixture</td></tr><tr><td>Lennox L LB Broth</td><td>Research Product International Corp.</td><td>L24066-5000.0</td><td>For making bacteria culture</td></tr><tr><td>Ampicillin sodium salt</td><td>Thermo Fisher Scientific</td><td>11593-019</td><td>Antibiotics</td></tr><tr><td>Kanamycin Sulfate</td><td>Thermo Fisher Scientific</td><td>15160-054</td><td>Antibiotics</td></tr><tr><td>Tetracycline</td><td>Thermo Fisher Scientific</td><td>64-75-5</td><td>Antibiotics</td></tr><tr><td>Gentamicin</td><td>Thermo Fisher Scientific</td><td>15710-064</td><td>Antibiotics</td></tr><tr><td>MAX Efficiency DH10Bac competent cells</td><td>Thermo Fisher Scientific</td><td>10361-012</td><td>For making bacmid DNA</td></tr><tr><td>S.O.C. Medium</td><td>Thermo Fisher Scientific</td><td>15544-034</td><td>For DNA transformation</td></tr><tr><td>CellFECTIN II Reagent</td><td>Thermo Fisher Scientific</td><td>10362-101</td><td>Insect cell transfection reagent</td></tr><tr><td>Bac-to-Bac Expression System</td><td>Thermo Fisher Scientific</td><td>10359-016</td><td>Baculovirus-insect cells expression kit</td></tr><tr><td>Bluo-gal</td><td>Thermo Fisher Scientific</td><td>15519-028</td><td>For isolation of recominant Bacmid DNA</td></tr><tr><td>IPTG</td><td>Thermo Fisher Scientific</td><td>15529-019</td><td>For isolation of recominant Bacmid DNA</td></tr><tr><td>pFastBac1</td><td>Thermo Fisher Scientific</td><td>10360014</td><td>Baculorirus expression vector</td></tr><tr><td>Sf9 cells</td><td>Thermo Fisher Scientific</td><td>11496015</td><td>Sf9 monolayer cells</td></tr><tr><td>Hi5 cells</td><td>Thermo Fisher Scientific</td><td>B85502</td><td>High Five insect cells</td></tr><tr><td>Grace&amp;rsquo;s insect medium, unsupplemented</td><td>Thermo Fisher Scientific</td><td>11595030</td><td>Sf9 cell transfection minimum medium</td></tr><tr><td>Grace&amp;rsquo;s insect medium, supplemented</td><td>Thermo Fisher Scientific</td><td>11605102</td><td>Sf9 monolayer cell culture complete medium</td></tr><tr><td>Sf-900 II SFM</td><td>Thermo Fisher Scientific</td><td>10902104</td><td>Sf9 suspension cell culture medium without FBS</td></tr><tr><td>Express Five SFM</td><td>Thermo Fisher Scientific</td><td>10486025</td><td>Hi5 cell culture medium</td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td>100 ml (10,000 I.U./ml)</td></tr><tr><td>L-Glutamine (200 mM)</td><td>Thermo Fisher Scientific</td><td>25030081</td><td>100 ml</td></tr><tr><td>FBS Certified</td><td>Thermo Fisher Scientific</td><td>16000-044</td><td>500 ml</td></tr><tr><td>6-well plates</td><td>Thermo Fisher Scientific</td><td>08-772-1B</td><td>Flat-bottom</td></tr><tr><td>150 mm plates</td><td>Thermo Fisher Scientific</td><td>353025</td><td>100/case</td></tr><tr><td>1.5 ml Microcentrifuge Tubes</td><td>USA Scientific</td><td>1415-2500</td><td>500 tubes/bag</td></tr><tr><td>15 ml conical screw cap centrifuge tubes</td><td>USA Scientific</td><td>1475-0511</td><td>25 tubes/bag</td></tr><tr><td>50 ml conical screw cap centrifuge tubes</td><td>USA Scientific</td><td>1500-1211</td><td>25 tubes/bag</td></tr><tr><td>Ni-NTA Superflow</td><td>Qiagen</td><td>30430</td><td>NiNTA resin</td></tr><tr><td>pH-indicator strips</td><td>EMD Millipore Corporation</td><td>1.09535.0001</td><td>pH 0 - 14</td></tr></tbody></table>

modifying baculovirus expression vectors to produce secreted plant proteins in insect cells

对科学家来说, 表达重组分泌真核蛋白用于结构和生物化学研究是一个挑战。杆状病毒介导的昆虫细胞表达系统是用于表达重组真核分泌蛋白的系统之一, 具有一定的转化后修饰。分泌蛋白需要通过分泌通路进行蛋白质糖基化、二硫化键形成和其他转化后的修饰。为改善现有分泌植物蛋白的昆虫细胞表达, 在启动子和多克隆点之间添加 GP67 或 hemolin 信号肽序列, 对杆状病毒表达载体进行修改。新设计的改进后的载体系统成功地生产出了拟南芥可溶性重组分泌植物受体蛋白的高产。对两种表达的植物蛋白, 拟南芥和 PRK3 膜受体的胞外域进行了结晶, 用于 x 射线晶体的研究。改进后的矢量系统是一种改良的工具, 可用于动物王国中重组分泌蛋白的表达。

如图 1所示, 两种改进的 pFastBac1 杆状病毒表达载体用于表达 GP67 或 hemolin 信号序列的分泌蛋白, 以取代目标基因的内在信号序列。病毒 GP67 和昆虫 hemolin 基因已被证明具有较高的分泌表达水平的细胞。融合蛋白与这两个信号序列中的任何一个有望大大提高分泌表达水平。在这两种改进的向量中, 都设计了相同的多个克隆站点 (MCS)。NotI 站点被蓄意放置?...

Watch this Scientific Journal Video about Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells at JoVE.com

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

改进杆状病毒表达载体生产昆虫细胞分泌植物蛋白

在这里, 我们提出了利用昆虫细胞和杆状病毒蛋白表达系统来产生大量植物分泌蛋白的蛋白质结晶的协议。用 GP67 或昆虫 hemolin 信号肽对昆虫细胞中植物蛋白分泌表达进行了改进, 并对杆状病毒表达载体进行了修正。

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

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Biochemistry

11.4K 次观看.  North Carolina State University. 在这里, 我们提出了利用昆虫细胞和杆状病毒蛋白表达系统来产生大量植物分泌蛋白的蛋白质结晶的协议。用 GP67 或昆虫 hemolin 信号肽对昆虫细胞中植物蛋白分泌表达进行了改进, 并对杆状病毒表达载体进行了修正。

视频: Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). However, a low expression level of foreign genes in insect cells was observed. Therefore, a transient gene expression system was constructed for improving protein expression. In this system, recombinant plasmids were constructed to contain the target sequence under the control of the Drosophila heat shock 70 (Dhsp70) promoter. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Furthermore, this system can be either transfected with one recombinant plasmid or co-transfected two potentially functionally antagonistic recombinant plasmids in insect cells. The protocol demonstrates the efficiency of this system and provides a practical case of this technique.

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

This protocol describes a heat shock-induced protein expression system (pDHsp/V5-His/sf9 cell system), which can be used for either expressing foreign proteins or evaluating the anti-apoptotic activity of potential foreign proteins and their truncated amino acids in insect cells.

昆虫细胞 (sf9) 外源基因在蛋白质功能检测中的瞬态表达

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

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遗传学

Production and Purification of Baculovirus for Gene Therapy Application

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a promising vector for gene therapy application. Here, baculovirus is produced by transient transfection of the baculovirus plasmid DNA (bacmid) in an adherent culture of Sf9 cells. Baculovirus is subsequently expanded in Sf9 cells in a serum-free suspension culture until the desired volume is obtained. It is then purified from the culture supernatant using heparin affinity chromatography. Virus supernatant is loaded onto the heparin column which binds baculovirus particles in the supernatant due to the affinity of heparin for baculovirus envelop glycoprotein. The column is washed with a buffer to remove contaminants and baculovirus is eluted from the column with a high-salt buffer. The eluate is diluted to an isotonic salt concentration and baculovirus particles are further concentrated using ultracentrifugation. Using this method, baculovirus can be concentrated up to 500-fold with a 25% recovery of infectious particles. Although the protocol described here demonstrates the production and purification of the baculovirus from cultures up to 1 L, the method can be scaled-up in a closed-system suspension culture to produce a clinical-grade vector for gene therapy application.

In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. The supernatant is purified by heparin affinity chromatography and further concentrated by ultracentrifugation. This protocol is useful for the production and purification of baculovirus for gene therapy application.

杆状病毒基因治疗应用的生产与纯化

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

哺乳动物 Bestrophin 离子通道的表达与纯化

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

生物化学

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

从新型H7N9流感病毒利用杆状病毒表达系统的功能重组血凝素和神经氨酸酶蛋白的表达

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

免疫与感染

Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. PRMTs can either mono- or dimethylate arginine guanidino groups symmetrically or asymmetrically. The enzymology of these proteins is a complex and intensely investigated area that requires milligram quantities of high-quality recombinant protein. The baculovirus expression vector system (BEVS) employing Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Spodoptera frugiperda 9 (Sf9) insect cells has been used for expression screening and production of many PRMTs, including PRMT 1, 2, and 4 through 9. To simultaneously screen for the expression of multiple constructs of these proteins, including domains and truncated fragments as well as the full-length proteins, we have applied scalable methods utilizing adjustable and programmable multichannel pipettes, combined with 24- and 96-well plates and blocks. Overall, these method adjustments enabled a large-scale generation of bacmid DNA, recombinant viruses, and protein expression screening. Using culture vessels with a high-fill volume of Sf9 cell suspension helped to overcome space limitations in the production pipeline for single batch large-scale protein production. Here, we describe detailed protocols for the efficient and cost-effective expression of functional PRMTs for biochemical, biophysical, and structural studies.

The baculovirus expression vector system (BEVS) is a robust platform for expression screening and production of protein arginine methyltransferases (PRMTs) to be used for biochemical, biophysical, and structural studies. Milligram quantities of material can be produced for the majority of PRMTs and other proteins of interest requiring a eukaryotic expression platform.

使用巴库洛病毒表达载体系统生产重组PRMT蛋白

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

在EMBL的MultiBac蛋白质复杂的生产平台

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

生物学

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

高级别瞬态表达重组蛋白的植物高效的农杆菌

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

优化和利用<em&gt;农杆菌</em&gt;介导的瞬态蛋白生产中<em&gt;烟草</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

细菌，昆虫细胞和植物系统:重组蛋白表达在不同的生物工厂的比较分析

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...